The Drosophila blastoderm embryo has provided key insights into eukaryotic transcriptional regulatory mechanisms, in particular the integration of basic biochemical processes in a developmental setting, where differential gene expression is used to drive the developmental fate of particular cells and tissues. Our research has focused on understanding the action of transcriptional repressors in this setting, because a major portion of the pattern generation relies on a cohort of zygotically expressed gap gene repressors. In the last grant period, we have utilized biochemical, genetic, and mathematical modeling approaches to understand the mechanisms of transcriptional repressors, to identify and characterize the corepressors that interact with these proteins, and quantitatively analyze the cis-regulatory code that describes the architecture of enhancers regulated by these proteins. These studies have demonstrated for the first time that short- and long-range repressors induce qualitatively very different effects at the chromatin level, and that the same set of corepressors can elicit very different effects depending on context. Our testing and modeling of short-range repressors revealed some of these contextual effects, providing the basis for a dissection of transcriptional "grammar" that unlocks genomic regulatory information. In our next funding period, we propose three aims to 1) extend our understanding of the cis-regulatory code through analysis and modeling of endogenous enhancers, 2) discern at a genomic level the activity and importance of multiple corepressors recruited by repressors, including distinct effects wrought by the mechanistically divergent short- and long-range repressors, and 3) characterize the importance in repression of a new type of corepressor, a DEAD box RNA helicase, which we have identified as a cofactor of the Knirps protein. The impact of these integrated aims will be to provide a detailed mechanistic and systems-wide understanding of conserved transcriptional regulatory processes that are used throughout metazoan development. The insights gained from these studies will provide essential underpinnings for population and evolutionary studies of development and disease in higher eukaryotes. PUBLIC HEALTH RELEVANCE: This research program seeks to understand the "language" of gene regulation that is encoded in the DNA. By determining how the proteins that serve as "off switches" act to interrupt gene expression, we will be able to identify processes that underlie animal development, and discern how they are misregulated in the context of disease. Furthermore, by use of modeling approaches, we will be able to better interpret genetic mutations that disrupt the integrity of the DNA switches to which these regulatory proteins bind.